Human parainfluenza virus type 1, 2, 3 and human respiratory syncytial virus (RSV) have been identified as the major viral pathogens responsible for severe respiratory tract infections in infants and young children (ref. 1 to 3--Throughout this specification, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately following the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). RSV has also been reported to cause significant morbidity in immunocompromised individuals and the elderly. Globally 65 million infections occur every year resulting in 160,000 deaths (ref. 4). In the USA alone, 100,000 children are hospitalized annually with severe cases of pneumonia and bronchiolitis resulting from an RSV infection (refs. 5, 6). Inpatient and ambulatory care for children with RSV infections has been estimated to cost in excess of $340 million each year in the USA (ref. 7). Severe lower respiratory tract disease due to RSV infection predominantly occurs in infants two to six months of age (ref. 8). The World Health Organization (WHO) and the National Institute of Allergy and Infectious Disease (NIAID) vaccine advisory committees have ranked RSV second only to HIV for vaccine development while the preparation of an efficacious PIV-3 vaccine is ranked in the top ten vaccines considered a priority for vaccine development. Both the annual morbidity and mortality figures as well as the staggering health care costs for managing paramyxoviridae infections including RSV have provided the incentive for aggressively pursuing the development of efficacious RSV vaccines.
RSV is a member of the Paramyxoviridae family of pneumovirus genus (ref. 2). The two major protective antigens of RSV are the envelope fusion (F) and attachment (G) glycoproteins (ref. 9).
In addition to the antibody response generated by the F and G glycoproteins, human cytotoxic T-cells have been shown to recognize the F protein RSV matrix (M) protein, nucleoprotein (N), small hydrophobic protein (SH) and nonstructural protein (lb) (ref. 10), produced following RSV infection. For PIV-3, the protective immunogen are the hemagglutinin-neuramidase (HN) protein and the fusion (F) protein.
Previous attempts to produce a safe and effective RSV vaccine were unsuccessful. Production of live attenuated RSV vaccines has had limited success. The mutants prepared to date have all been either over-attenuated, virulent or genetically unstable. A formalin-inactivated (FI) RSV vaccine developed in the 1960's failed to provide adequate protection in clinical trials (refs. 8, 11, 12). In fact, immunization of seronegative infants with the FI-RSV vaccine resulted in the exacerbation of RSV disease (immunopotentiation) in some vaccinees following exposure to wild type virus. Identification of the major immunoprotective antigens of RSV has provided the scientific rationale for pursuing a subunit approach to RSV vaccine development. However, efficacy of the RSV subunit vaccines tested to date have been inconsistent (ref. 12). There is also conflicting reports in the literature on the ability of an alum-adjuvanted RSV vaccine containing the F protein purified from virus infected cells by immunoaffinity chromatography (PFP-1) to cause enhanced pulmonary pathology (immunopotentiation) following live virus challenge (ref. 13 and 14). There is a definite requirement for the development of a safe and efficacious RSV vaccine.
One of the main obstacles in developing a safe and effective RSV vaccine has been to produce a vaccine formulation that can elicit a protective immune response without causing exacerbated disease. Elucidation of the mechanism(s) involved in the potentiation of RSV disease is important for the design of safe RSV vaccines, especially for the seronegative population. Recent experimental evidence suggests that an imbalance in cell-mediated responses may contribute to immunopotentiation (ref. 15). Enhanced histopathology observed in mice that were immunized with the FI-RSV and challenged with virus could be abrogated by depletion of CD4+ cells or both interleukin-4 (IL-4) and IL-10 (ref. 16). Experimental results indicated that induction of a Th-2 type response may play a role in disease potentiation. BALB/c mice given live virus intranasally or intramuscularly elicited a Th-1 type response, whereas FI-RSV induced a Th-2 type of response. These results were recently substantiated by the finding that BALB/c mice that were immunized with the FI-RSV vaccine had a marked increase in the expression of mRNA (from cells in the bronchoalveolar lavage fluid) for the Th-2 cytokines IL-5 and IL-13 (ref. 17).
Studies on the development of live viral vaccines and glycoprotein subunit vaccines against parainfluenza virus infection are being pursued. Clinical trial results with a formalin-inactivated PIV types 1,2,3 vaccine demonstrated that this vaccine was not efficacious (refs. 33, 34, 35). Further development of chemically-inactivated vaccines was discontinued after clinical trials with a formalin-inactivated RSV vaccine demonstrated that not only as the vaccine not effective in preventing RSV infection but many of the vaccinees who later became infected with RSV suffered a more serious disease. Most of parainfluenza vaccine research has focussed on candidate PIV-3 vaccines (ref. 36) with significantly less work being reported for PIV-1 and PIV-2. Recent approaches to PIV-3 vaccines have included the use of the closely related bovine parainfluenza virus type 3 and the generation of attenuated viruses by cold-adaptation of the virus (refs. 37, 38, 39, 40).
Another approach to parainfluenza virus type 3 vaccine development is a subunit approach focusing on the surface glycoproteins hemagglutinin-neuraminidase (HN) and the fusion (F) protein (refs. 41, 42, 43). The HN antigen, a typical type II glycoprotein, exhibits both haemagglutination and neuraminidase activities and is responsible for the attachment of the virus to sialic acid containing host cell receptors. The type I F glycoprotein mediates fusion of the viral envelope with the cell membrane as well as cell to cell spread of the virus. It has recently been demonstrated that both the HN and F glycoproteins are required for membrane fusion. The F glycoprotein is synthesized as an inactive precursor (F) which is proteolytically cleaved into disulfide-linked F2 and F1 moieties. While the HN and F proteins of PIV-1, 2 and 3 are structurally similar, they are antigenically distinct. Neutralizing antibodies against the HN and F proteins of one PIV type are not cross-protective. Thus, an effective PIV subunit vaccine must contain the HN and F glycoproteins from the three different types of parainfluenza viruses. Antibody to either glycoprotein is neutralizing in vitro. A direct correlation has been observed between the level of neutralizing antibody titres and resistance to PIV-3 infections in infants. Native subunit vaccines for parainfluenza virus type 3 have investigated the protectiveness of the two surface glycoproteins. Typically, the glycoproteins are extracted from virus using non-ionic detergents and further purified using lectin affinity or immunoaffinity chromatographic methods. However, neither of these techniques may be entirely suitable for large scale production of vaccines under all circumstances. In small animal protection models (hamsters and cotton rats), immunization with the glycoproteins was demonstrated to prevent infection with live PIV-3 (refs. 44, 45, 46, 47, 48). The HN and F glycoproteins of PIV-3 have also been produced using recombinant DNA technology. HN and F glycoproteins have been produced in insect cells using the baculovirus expression system and by use of vaccinia virus and adenovirus recombinants (refs. 49, 50, 51, 52, 53). In the baculovirus expression system, both full-length and truncated forms of the PIV-3 glycoproteins as well as a chimeric F-HN fusion protein have been expressed. The recombinant proteins have been demonstrated to be protective in small animal models (see WO91/00104, U.S. patent application Ser. No. 07/773,949 filed Nov. 29, 1991, assigned to the assignee hereof).
The construction of the recombinant poxviruses is described in a number of granted United States patents, including U.S. Pat. Nos. 4,603,112, 4,769,330, 4,722,848 and 5,110,587 relating to various recombinant virus constructs, U.S. Pat. Nos. 5,453,364, 5,225,336 and 5,155,020 relating to attenuated recombinant vaccinia virus constructs and U.S. Pat. No. 5,174,993 relating to recombinant avipox virus constructs. The disclosure of these patens are incorporated herein by reference.
Live recombinant poxviruses expressing the relevant viral proteins may be used alone or as priming immunogens in a prime/boost regime with the subunit vaccine. Despite having promising attributes as a "universal" immunization vehicle, safety issues have provided a concern for the re-introduction of vaccinia virus as an immunizing agent. These concerns stem from complications observed during the Smallpox Eradication Program (ref. 18). From one perspective, the safety issues surrounding the use of vaccinia-based vaccine candidates have been addressed with the development of the NYVAC and ALVAC vectors.
The NYVAC strain was derived from the vaccinia virus Copenhagen strain by the precise deletion of 18 ORFs encoding functions implicated in the pathogenicity of orthopoxviruses, as well as host-range regulatory functions governing the replication competency of these viruses on cells from certain species (ref. 19). General biological properties of NYVAC include: [1] a highly debilitated replicative capacity on cells derived from mice, swine, equids, and humans; [2] the ability to replicate with wildtype efficiency on primary chick embryo fibroblasts, and [3] a highly attenuated phenotype in immunocompetent and immunocompromised animal systems used historically to assess the virulence of vaccinia virus strains (ref. 19). Despite these highly attenuated properties, NYVAC has been shown to function effectively as an immunization vehicle (ref. 19, 20). These properties are consistent with NYVAC providing a safer alternative to existing vaccinia virus vaccine strains for developing vector-based vaccine candidates. Due to the attenuation profile of NYVAC, the Recombinant DNA Advisory Committee of the National Institutes of Health has reduced the biological containment level of this virus from BSL-2 to BSL-1. It is the only member of the Orthopoxvirus genus accorded to a BSL-1 biocontainment level.
The basic vaccinia virus vector technology has been extended to other members of the poxvirus family. Extension to the Avipoxvirus genus, in particular fowlpoxvirus (FPV), was targeted for species-specific applications in the poultry industry (ref. 21). Studies with a FPV recombinant expressing an immunogen from a mammalian pathogen (the rabies virus glycoprotein G), however, demonstrated the ability of this recombinant to elicit immune responses in a number of non-avian species (ref. 22), thus establishing these viruses as viable candidates for developing non-replicating vector-based vaccine candidates for veterinary and human application. The inability of the Avipoxviruses to productively replicate in non-avian species provides an exquisite safety barrier against the occurrence of vaccine-associated and vaccine-induced complications.
Subsequent studies with canarypoxvirus (CPV)-based recombinants in non-avian species also demonstrated their utility as immunization vehicles (refs. 23, 24). In this regard the canarypoxvirus-based recombinants were found superior to similar FPV recombinants and equivalent to thymidine-kinase mutants of replication-competent vaccinia virus recombinants (refs. 19, 24). A plaque-cloned isolate of CPV was derived from the vaccine strain Kanapox and designated ALVAC (ref. 19).
ALVAC, like NYVAC, has demonstrated a highly attenuated phenotype in a number of animal systems comparing existing vaccinia virus vaccine strains (ref. 19). The Recombinant DNA Advisory Committee has reduced the biological containment for ALVAC to BSL-1. Furthermore, ALVAC has been shown to be an effective immunization vehicle in a number of non-avian species including humans (ref. 25). The concept of using a non-replicating vector in humans was supported by the results of phase I clinical trials using an ALVAC-based rabies G (ref. 26) and an ALVAC-HIV-1.sub.MN env (ref. 27) recombinant. In each study, the ALVAC-based recombinant elicited antigen-specific immune responses the heterologous antigen. Thus, the use of either ALVAC or NYVAC recombinants expressing the pertinent RSV proteins represents a promising approach for RSV vaccine development.